| Objective: Hepatocellular carcinoma(HCC)accounts for 90% of primary liver cancer cases worldwide.Given its rapid progression,these patients are prone to postoperative recurrence and distant metastasis with poor prognosis.New potential targets that can affect the progress and metastasis of HCC must be identified.Molecular chaperones are critical mediators of oncogenesis and necessary for cell survival.The cytoplasmic chaperone TRi C(T-complex protein-1 ring complex,also known as CCT)comprises two back-to-back stacked rings,with each ring containing eight subunits(CCT1 to CCT8).Furthermore,a single CCT subunit also has a protein folding ability and independent function.However,the roles of the CCT1 subunit(also known as TCP1)in HCC and its molecular mechanism have not been investigated thoroughly.Understanding its role in tumor development and progression and its mechanism can provide new ideas and strategies for treating HCC.Methods:(1)The expression of TCP1 in HCC and its relationship with the survival and prognosis of patients by using data from clinical specimens and the public databases(TCGA and Oncomine).(2)Hepatoma cell lines with a decreased expression of TCP1 were constructed through lentivirus,which were then observed the changes in its proliferation,migration,cell cycle process by CCK8 assay,transwell and FCM(Flow cytometry).Xenograft models to observe tumor growth and lung metastasis in vivo.(3)A high-throughput whole genome expression chip was used to analyze the gene expression difference between sh-TCP1(knockdown of TCP1)and NC(negative control).WB was used to detect the expression of key molecules in the Wnt signaling pathway targeting Wnt7b.(4)Promo and Jaspar databases predict the transcription factors involved in the regulation of the WNT7B promoter.Several luciferase reporter plasmids were constructed based on the predictions and to determine whether P53 regulate WNT7B transcription by the double luciferase report analysis.Then mutant plasmids were constructed to analyse P53 binding site on the WNT7B promoter,and the ChIP assay to further verification.The CoIP to analyse the protein–protein interaction between TCP1 and P53.Results:(1)The level of TCP1 in HCC was significantly higher than that in the corresponding adjacent tissues.Meanwhile,compared with patients with a low expression of TCP1,those patients with a high expression of TCP1 had a poor survival and prognosis.(2)the downregulation of TCP1 can significantly reduce cell viability,inhibite cell proliferation and migration,the cell cycle was blocked in the G2/M phase in vitro.In addition,xenograft models indicated that the knockdown of TCP1 inhibited the growth and lung metastasis in vivo.(3)The KEGG pathway analysis showed that the Wnt signaling pathway was the most important.Western blot showed that the downregulation of TCP1 significantly increased the expression of p-β-catenin and decreased expression of Wnt7b and β-catenin,the expression of E-cadherin were significantly upregulated,whereas vimentin,c-Myc,and cyclin-D1 were significantly downregulated.After treatment with SKL2001(a Wnt pathway agonist targeting β-catenin),the changes in the related protein levels due to TCP1 knockdown were significantly reversed.(4)The P53 expression significantly enhanced the luciferase activities induced by the WNT7B promoter,and P53 binding active sites in the WNT7B promoter region(-390 nt ～-383nt).TCP1 knockdown significantly inhibits the transcriptional regulation of P53 on WNT7B and there is a protein–protein interaction between TCP1 and P53.Conclusions:(1)TCP1 expression is elevated in HCC tissues and correlates with a poor prognosis.(2)Knockdown of TCP1 inhibits the proliferation and migration of HCC in vitro and the growth and lung metastasis of hepatoma cells in vivo.(3)Downregulated TCP1 expression inhibits the Wnt/β-catenin signaling pathway of HCC cells through Wnt7b.(4)TCP1 regulates WNT7B transcription by binding transcription factor P53 in HCC cells... |
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